Lysozyme In Presence Research Articles

Exploring the underlying mechanism of interaction of Sulindac with Human hemoglobin and Lysozyme: Multi-spectroscopic, molecular docking, DFT and MD simulation approaches

Contrasting behavior of Nonsteroidal anti-inflammatory drug Sulindac (SDC) towards two different proteins Human hemoglobin (HHb) and Lysozyme (LYS) has been explored by multispectroscopy, molecular docking, DFT and MD Simulation approaches. The fluorescence quenching experiments showed that SDC strongly binds with both proteins in a static manner. Alteration in the secondary structure of HHb and LYS in presence of SDC was further investigated by CD and UV–visible spectroscopy. It is observed that on addition of SDC, the % α-helix of native HHb decreases whereas it increases for LYS-SDC system substantiated the fact SDC stabilized the secondary structure of LYS and destabilized the HHb structure thus, showing the contrasting traits. Experimental results further validated by computational techniques. Molecular docking and DFT were conducted to study the binding sites, nature of interactions and HOMO-LUMO energy gap in both the systems which revealed that the binding of SDC with both proteins resulted in the considerable change in the molecular structure of the former. Additionally, we performed MD simulations to study the structural stability of Lysozyme and Hemoglobin, both unbound and bound to the Sulindac molecule. Sulindac binding showed distinct impacts on protein stability in both proteins, supported by global and essential dynamics.This study not only provides important insights into the binding of SDC with HHb and LYS but also it provides a useful direction in the pharmacology and clinical medicine fields.

Probing the interaction and aggregation of lysozyme in presence of organophosphate pesticides: a comprehensive spectroscopic, calorimetric, and in-silico investigation

Organophosphorus pesticides (OPs) are widely used in agriculture and may contaminate food or water, leading to potential health risks. However, there are few reports on the effect of OPs on protein conformation and aggregation. Hence, in this paper, we have characterized the impact of two OPs, chlorpyrifos (CPF) and methyl parathion (Para), on the model protein HEWL using biophysical and computational methods. The steady-state and time-resolved spectroscopy, Circular dichroism (CD), molecular dynamics simulation, and isothermal titration calorimetry were employed to investigate the binding interactions between HEWL and OPs. The steady-state and time-resolved fluorescence spectroscopy confirm the presence of both static and dynamic quenching between OPs and proteins. Based on fluorescence, MD, and CD results, it was found that the OPs not only show strong binding but also destabilize the protein structure and alter the secondary and tertiary structure of the protein. The molecular docking results showed that OPs entered the binding pocket of the HEWL molecule and interacted through hydrophobic and hydrogen bond interactions. The thermodynamic studies indicated that the binding was spontaneous and OPs have shown an effect on the aggregation process of HEWL. Finally, the protein aggregation process was studied using fluorescence and SDS-PAGE studies in the presence of both the OPs and found to enhance the aggregation process in the presence of OPs. These results provide insights into the potential health risks associated with OPs and highlight the importance of understanding their interactions with biological macromolecules. Communicated by Ramaswamy H. Sarma

Interactions of lysozyme with hydrophobic and hydrophilic non-steroidal anti-inflammatory drugs: Spectroscopic and molecular docking analyses

Non-covalent molecular interaction of lysozyme with two very important non-steroidal anti-inflammatory drugs (paracetamol or acetaminophen and ibuprofen) were studied using fluorescence, UV–visible and circular dichroism spectroscopies. In addition, the interaction was also studied using molecular docking simulations. Changes in the difference UV–visible absorption spectra of lysozyme in presence of the drugs were the indication of the complex formation between drug and the protein. Fluorescence spectroscopic observations showed that the fluorescence intensity of lysozyme initially increased on the addition of paracetamol then start to decrease at higher concentrations of the latter, however, a constant increase in the fluorescence intensity of the protein was observed on the addition of ibuprofen. The irregular pattern observed in case of paracetamol was ascribed to the inner filter effect, which after correction turned into the uniform fluorescence enhancement. Both drugs have shown a good interaction with lysozyme, although, the interaction of ibuprofen was slightly stronger than the one with paracetamol. The binding of paracetamol with lysozyme was taken place with contribution of both hydrophobic forces as well as hydrogen bodings whereas in lysozyme-ibuprofen case, hydrophobic interactions were the dominating forces. Paracetamol as well as ibuprofen induced the secondary structure of lysozyme by increasing the α-helicity of the protein. Results obtained through molecular docking were in excellent agreement with the one obtained through experimental studies.

Probing the interaction of cephalosporin antibiotic “cefoperazone” with lysozyme using spectroscopic and in silico methods: Effect of paracetamol on binding

The interaction of lysozyme with cefoperazone was studied by means of spectroscopic and computational approaches. The change in the UV–visible spectrum of lysozyme in presence of cefoperazone was an indication of the complex formation between them. Fluorescence spectroscopy suggested that there was a fair interaction between the protein and drug which was taken place via dynamic quenching mechanism and the binding ratio was approximately 1:1. The binding was energetically feasible and principally supported by the hydrophobic forces. CD spectroscopic studies have shown that cefoperazone induced the secondary structure of lysozyme by increasing the α-helical contents of the latter. In silico studies revealed that the large nonpolar cavity was the preferred binding site of cefoperazone within lysozyme and the interaction was taken place mainly through hydrophobic forces with small involvement of hydrogen bonding and electrostatic interactions which is in good agreement with the experimental analyses. Effect of paracetamol was also seen on the binding and it was found that paracetamol had a negative influence on the binding between cefoperazone and lysozyme.

Thermodynamics of modulation of interaction of α-helix inducer 2, 2, 2-trifluoroethanol with lysozyme in presence of cationic, anionic and non-ionic surfactants

The interactions of anionic sodium dodecyl sulphate (SDS), cationic cetyltrimethylammonium bromide (CTAB) and nonionic triton X-100 (TX-100) surfactants with lysozyme at pH = 2.4 have been studied individually as well as in combination with 2,2,2-trifluoroetanol (TFE). Urea has also been used in combination with surfactants. By using these combinations, efforts have been made to obtain partially folded conformations of the protein in the presence of surfactants and effect of α-helix inducer 2,2,2-trifluoroethanol on these intermediate states. Thermodynamic analysis of all these interactions has been done employing a combination of UV-visible, fluorescence and circular dichroism spectroscopies. The results have been correlated with each other and characterized qualitatively as well as quantitatively. At lower concentration of surfactant, the thermodynamic parameters indicated the destabilizing effect of SDS, stabilizing effect of CTAB and unappreciable destabilizing impact of TX-100 on lysozyme. The enhancement in destabilization effect or reduction in stabilization effect of surfactants on lysozyme in the presence of TFE and urea has also been indicated. Communicated by Ramaswamy H. Sarma

Modulation of drug binding ability and augmented enzymatic activity of lysozyme stabilized in presence of surface-active ionic liquids

Binding of polyphenolic compounds like genistein (GTN) and ellagic acid (EGA) with lysozyme (LYZ) shows strong modulation in presence of surface-active ionic liquids (SAILs), N-dodecylpyridinium bromide(PYD),N-dodecyl-N-methylmorpholinium bromide (MOR), 1-dodecyl-3-methylimidazolium bromide(IMD). Fluorescence of both the native LYZ and LYZ-SAIL complex quenched in presence of the drugs. Both GTN (15.62 × 104 M−1) and EGA (53.70 × 104 M−1) show strong binding to LYZ in presence of IMD in comparison with native LYZ (8.18 × 104 M−1 and 31.28 × 104 M−1, respectively). Stern-Volmer (SV) quenching constant (KSV) of GTN binding to LYZ-PYD complex is almost fourfold (35.79 × 104 M−1) higher than the corresponding values in native LYZ. Spectral response of amide I and amide II bands of LYZ remains same even in presence of SAILs, advocating the intactness of the secondary structure of LYZ. MD simulation studies indicate that the drug molecules preferentially bind near some key tryptophan residues through pi-stacking and hydrophobic interactions. Non-specific interactions of SAILs with the protein seem to modulate the conformational flexibility of the protein, which affects the protein-drug binding affinity. Considerably higher activity of LYZ against Gram positive bacteria Micrococcus lysodeikticus was also observed in presence of IMD. Significant modulation in binding behavior in presence of SAIL indicates that the drug transportation capacity of LYZ can be controlled even in the pre-micellar concentration and could further be exploited in advanced drug delivery techniques.

Binding studies for the interaction between hazardous organophosphorus compound phosmet and lysozyme: Spectroscopic and In-silico analyses

Organophosphorus compounds are generally toxic compounds found uses in agriculture, pharmaceutics, and other industries. These organophosphorus compounds are life threatening and may cause mortalities on acute exposure. Also, some of these are well known nerve agents. Phosmet is one such organophosphorus compound with moderate nerve toxicity on prolonged exposure. Phosmet is widely used pesticide on various trees including apple, pear, peach, almond and vines like grape vine. Thus, phosmet is the major contaminant of most of the consumed fruits.The present study investigated the binding of phosmet with lysozyme to understand the toxicology and the transport of phosmet using various spectroscopic techniques, molecular modeling and enzymatic assay. The ability of lysozyme to act as a biomarker for phosmet can also be revealed by the current study. The UV–Visible spectrum of lysozyme in presence of phosmet indicated hyperchromicity in the absorbance peak of lysozyme. The fluorescence quenching of lysozyme in presence of phosmet involve predominantly static quenching. The complexation is further quantified in terms of a binding constant (Kb) and the number of sets of equivalent binding sites (n) with the help of double logarithmic regression curve. The Kb value of 103–104 M−1 and n value near 1 indicated that phosmet is binding through non-covalent forces in single set of equivalent binding sites. Static quenching was additionally confirmed by modified Stern-Volmer plot for lysozyme-phosmet interactions. Post binding protein conformation changes and the enhanced protein stability of lysozyme were showed by CD spectrum and melting curves of lysozyme. The FT-IR spectrum also revealed the changes in secondary structure content of protein on interaction with phosmet. The docked structure for binding of phosmet with lysozyme were achieved using molecular docking analysis with a glide score −5.273 k J/mol-1 that indicated towards moderate binding as suggested by other experimental studies. The enzymatic activity of lysozyme depicted an increase of 16% after binding with phosmet indicative of interaction with the active site.

Interaction of Carrier Protein with Potential Metallic Drug Candidate N-Glycoside 'GATPT': Validation by Multi-Spectroscopic and Molecular Docking Approaches.

Lysozyme is often used as a model protein to study interaction with drug molecules and to understand biological processes which help in illuminating the therapeutic effectiveness of the drug. In the present work, in vitro interaction studies of 1-{(2-hydroxyethyl)amino}-2-amino-1,2-dideoxy-d-glucose triphenyl tin (IV) (GATPT) complex with lysozyme were carried out by employing various biophysical methods such as absorption, fluorescence, and circular dichroism (CD) spectroscopies. The experimental results revealed efficient binding affinity of GATPT with lysozyme with intrinsic binding (Kb) and binding constant (K) values in the order of 105 M−1. The number of binding sites and thermodynamic parameters ΔG, ΔH, and ΔS at four different temperatures were also calculated and the interaction of GATPT with lysozyme was found to be enthalpy and entropy driven. The CD spectra revealed alterations in the population of α–helical content within the secondary structure of lysozyme in presence of GATPT complex. The morphological analysis of the complex with lysozyme and lysozyme-DNA condensates was carried out by employing confocal and SEM studies. Furthermore, the molecular docking studies confirmed the interaction of GATPT within the larger hydrophobic pocket of the lysozyme via several non-covalent interactions.

Behavior of lysozyme within ionic liquid-in-water microemulsions

Ionic liquids (ILs) and IL-based microemulsions are of immense importance due to their broader application potential in various interdisciplinary research areas such as, synthesis, catalysis, biochemical processes, pharmaceutics, and material science. In this manuscript, we have studied the behavior of an antimicrobial enzyme lysozyme within IL-in-water (IL/W) microemulsions formed by hydrophobic IL 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF6] in nonionic surfactant Brij-35 solution. It is noteworthy that the fluorophores and chromophoric responses clearly indicate significant changes in the micropolarity and microviscosity within aqueous Brij-35 micellar solution in the presence of IL [bmim][PF6] forming microemulsions. DLS shows the size of these nanostructures formed are of 6–8 nm range. IL-lysozyme interactions within the IL/W microemulsions were studied using UV–Vis, steady-state and time-resolved fluorescence and FT-IR spectral investigations. Increase in Brij-35 concentration increases the lysozyme fluorescence intensity in absence of the IL, whereas increase in IL concentration within the IL/W microemulsions results in gradual decrease in the lysozyme fluorescence intensity as a results of IL-lysozyme binding. Both steady-state and time-resolve fluorescence results suggest fluorescence quenching of lysozyme in presence of IL [bmim][PF6] within both aqueous solution and Brij-35 IL/W microemulsions. Further, electrochemical response from cyclic voltammetric studies also confirms the binding of lysozyme with IL [bmim][PF6]. FTIR spectral responses show that lysozyme remains stable within the IL/W microemulsions amid IL-lysozyme binding observed from UV–Vis and fluorescence studies.

Intramolecular OH stretching analysis of hydrated lysozyme in presence of trehalose by IR spectroscopy

The present work reports the analysis of the intramolecular OH stretching band obtained by InfraRed spectroscopy measurements. In order to characterize the effect of trehalose on the hydration properties of lysozyme the so-called two-state model is adopted for the analysis of the intramolecular OH stretching band. This latter assumes that, provided that the trehalose OH stretching contribution is subtracted, water molecules can be partitioned into two different states of inter-molecular bonding: molecules with two OH groups both hydrogen-bonded within a tetrahedral network, and molecules with one or two dangling OH groups. What emerges from this study is that trehalose significantly influences the hydrogen bond network of water and its temperature behaviour. Such a result confirms that the trehalose induced strengthening of the hydrogen-bond network leads to a stabilization of the lysozyme structure. Moreover, the analysis of the spectra temperature dependence shows a trehalose-induced higher thermal restraint of the lysozyme-trehalose-water system in respect to the lysozyme-water mixture.

Binding Interaction of Juglone with Lysozyme: Spectroscopic Studies Aided by In Silico Calculations

Binding interactions between the drug Juglone (JUG) and Lysozyme (LYZ) have been explored in details from spectroscopic studies aided by in silico calculations. UV–Vis, steady state and time resolved fluorescence spectroscopic studies indicate the formation of LYZ–JUG complex in the ground state. Quenching of corrected fluorescence spectra of LYZ in presence of JUG at varied concentrations in different temperature range have been estimated from Stern–Volmer (SV) plots. Time resolved fluorescence spectroscopic studies confirm the mechanism of quenching to be of static type. Binding constant associated with the LYZ–JUG complex has been estimated from Scatchard plot. The number of binding sites, thermodynamic parameters and the modes of interaction are also estimated. Synchronous fluorescence spectra monitored at two discrete wavelength windows confirm the prominent role of Tryptophan residues towards quenching of fluorescence in LYZ. The circular dichroism (CD) spectra signify alterations in the population of α–helical content within the secondary structure of LYZ in presence of JUG molecules. REES of LYZ in the presence of JUG further signify definite impact of the drug JUG molecule on the Trp residues of the protein. The experimental observations are supported by in silico molecular docking and molecular dynamics simulations.

Effect of physical interactions on structure of lysozyme in presence of three kinds of polysaccharides.

In this work the influences of κ-carrageenan (CRG), konjac glucomannan (KGM) and inulin on lysozyme (Ly)'s structure, activity, and their complex phase behavior were investigated through spectroscopy and activity measurement in heated and unheated conditions. It was found that the impact on the structure and activity of Ly was determined by the interactions with polysaccharides. After heat treatment, KGM and CRG improved the stability of complex systems. However, inulin did not have significant impact. Heating process promoted to change the structure of Ly, and the intervention retard following the sequence of CRG > KGM > inulin. The worthwhile work indicated protein's structure and activity could be regulated by the interaction with polysaccharide, which might provide theoretical basis for food preservation and processing in different temperature treatments. Besides, the bidirectional effects of polysaccharide on protein would be beneficial to rational selection of functional properties of polysaccharide/protein systems.

Interaction of caffeine and sulfadiazine with lysozyme adsorbed at colloidal metal nanoparticle interface: influence on drug transport ability and antibacterial activity

The modulated bioactivity of proteins immobilized on nanoparticle (NP) interfaces is of tremendous interest toward designing better therapeutic and diagnostic tools. In this work, binding behavior and the antibacterial activity of free lysozyme (LYS) as well as its non-covalent assembly with silver (Ag) and gold (Au) colloidal NPs were compared in presence of two model drugs, viz. sulfadiazine (SDZ) and caffeine (CAF). Intrinsic protein fluorescence was found to quench due to the formation drug–protein complex in case of CAF resulting a linear Stern–Volmer (SV) plot with KSV = 1.83 × 103 M−1.On the other hand, a positive deviation beyond [SDZ] ~0.15 mM is explained due to the formation of a fluorophore – quencher sphere with radius of 13.85 ± 1.80 Å that results almost one order of magnitude higher KSV (1.75 × 104 M−1). Molecular docking calculation also predicts relatively more stabilized complex of SDZ with LYS in comparison to CAF (ΔE ~ 3 kJ mol−1). Synchronous fluorescence results corresponding to Trp and Tyr residues as well as FTIR spectra in the amide I region of LYS confirms minimal deformation in the LYS secondary structure on adsorption to spherical NP surface. Although the nature of LYS–drug interaction remains invariant, the extent of quenching interaction as well as the drug binding ability is strongly modulated in presence of NPs. Further, the antibacterial activity of LYS in presence of the investigated drugs shows 9–14% upsurge with AuNP, in sharp contrast to ca. 31–34% decrease in AgNP.

Characterization of heat induced spherulites of lysozyme reveals new insight on amyloid initiation.

Here, we report results obtained during our experiments to visualize how heat transforms globular protein, lysozyme into building block of β-amyloids. Light scattering experiments showed formation of lower order associated species around 50–70 °C followed by rapid cooperativity to β-amyloid fibrils. Interestingly, crystallization drops set at higher temperatures either led to aggregates or spherulites. The latter possess an amorphous β-fibril rich core with thin crystalline needles projecting outwards. Diffraction of the crystalline outgrowths revealed novel dimers and trimers of lysozyme where individual chains were similar to monomer with marginal gain in β-sheet content. Importantly, analysis of Amide I stretching frequencies showed that protein loses its secondary structure at temperatures higher than where we obtained crystals followed by rapid gain in β-sheet content. Interestingly, attempts to use the needles as seeds for more crystals led to “broom-like” fibril formations at the ends. Further, aggregation inhibitors like arginine and benzyl alcohol completely obliterated spherulites formation during crystallization. Refinement of crystals of lysozyme in presence of these molecules showed these small molecules bind to the interfaces of heat associated dimers and trimers. Overall our work concludes that heat induced weakly associated structures of lysozyme are the first step towards its amyloid formation.

Fluorescence behavior of globular proteins from their bulk and thin film conformations in presence of mono-, di- and tri-valent ions

Photoluminescence behavior of globular proteins, lysozyme and bovine serum albumin (BSA), from their bulk and thin film conformations have been studied in presence of mono-, di- and tri-valent ions by using fluorescence and UV-Vis spectroscopy at two different temperatures and the morphology of the protein thin films have been studied by using atomic force microscopy. Protein- and ion-dependent dynamic and static quenching behaviors have been identified. While dynamic quenching is observed for lysozyme for all the three different valent ions, BSA shows no quenching for mono-valent (Na+) ions, dynamic quenching for di-valent (Ni2+) ions and static quenching for tri-valent (Fe3+) ions at pH≈5.5. After heat treatment, as the conformation of the protein molecules changes, the quenching efficiency for lysozyme in presence of ions decreases but shows enhancement for BSA. In thin film geometry, the molecular conformation of both lysozyme and BSA modifies on the solid surfaces and hence quenching efficiency also modifies in comparison with that of bulk and as a result the quenching efficiency for lysozyme increases but decreases for the BSA film.

Protein interactions of Merocyanine 540: Spectroscopic and crystallographic studies with lysozyme as a model protein

Spectroscopic and crystallographic studies reveal that Merocyanine 540 (MC 540), a well-known therapeutically important anionic cyanine dye, interacts with hen egg white lysozyme in ground state. The formation of the complex is validated by two isosbestic points in absorption spectra of lysozyme with varied concentration of MC 540 and appearance of an isodichroic point in induced CD spectra of MC 540 with lysozyme. The blue shift of fluorescence maximum of lysozyme in presence of MC 540 shows hydrophobic effect on Trp due to complex formation probably through cooperative binding. Above 1:3M stoichiometric ratio (lysozyme:MC 540) an additional fluorescence hump arises because of structural changes in protein, where MC 540 acts as self-denaturant, inducing non-linearity in Stern–Volmer plot. The van’t Hoff isotherms with negative changes in enthalpy at lower concentration and positive changes in entropy for entire concentration range of MC 540 depict the binding forces as hydrogen bonding/van der Waal’s and ionic/hydrophobic respectively. Finally X-ray crystallographic structure of the complex shows that MC 540 adopts two conformations, cis and trans, while it binds to lysozyme. Benzoxole moiety of MC 540 interacts with Trp123 through π-stacking and SO32- group is stabilized by ionic interaction/H-bonding with Arg125 of lysozyme.

Identification and Characterization of Molten Globule-Like State of Hen Egg-White Lysozyme in Presence of Salts Under Alkaline Conditions

In the present study, we elucidated the effect of potassium salts on alkali denatured hen egg white lysozyme (EC 3.2.1.17) using intrinsic/extrinsic fluorescence as well circular dichroism (CD) spectroscopic methods. Intrinsic fluorescence studies revealed that various potassium salts mediate stabilization of lysozyme against alkali denaturation. Far and near UV CD spectrum studies, showed that 2M KCl induced appreciable amount of secondary structure with minimum tertiary contacts in lysozyme at pH 12.6. Acrylamide quenching studies suggest that at pH 12.6, the presence of 2M KCl causes reduced accessibility of the quencher to tryptophan residues of the protein presumably because of its compact conformation. In summary, the results of present study suggest that lysozyme attains a compact folded intermediate with molten globule like characteristics at alkaline pH in presence of potassium chloride.

Protoplast fusion: a tool for intergeneric gene transfer in bacteria.

Protoplasts can be isolated from bacterial cells by digestion of the cell wall with the help of lysozyme in presence of osmotic stabilizers. Fusion of protoplasts can be induced by chemical fusogens like polyethylene glycol. The electrofusion technique has been reported in bacteria in which the fusion frequency is much higher than that obtained by PEG induced protoplast fusion. This technology allows recombination to take place not only between related species but also between unrelated genera and is of great potential in the breeding and improvement of industrial strains. This review includes the information and developments on the protoplast fusion in bacteria with special reference to genetic recombination by protoplast fusion between phylogenetically unrelated bacteria.

On the Mechanism of the Acridine Orange Sensitized Photodynamic Inactivation of Lysozyme II. Kinetics in Presence of N-Acetylglucosamine

The photodynamic deactivation of lysozyme in presence of acridine orange is caused by a reaction between singlet oxygen formed via the dye triplet state and the protein. In order to identify the region where the singlet oxygen reacts with the protein we have investigated the kinetics of the deactivation in presence ofthe inhibitor of the enzymatic reaction N-acetylglucosamine (GlcNAc). The overall experimental rate constant becomes slower with increasing saccharide concentrations. As we can exclude experimentally that this kinetical effect is caused in presence of the saccharide by a physical quenching of singlet oxygen or of the dye triplet state it has to be assumed that GlcNAc protects the surrounding of its bindings place at subsite C of the enzymatic center sterically against an attack of singlet oxygen. In this region three tryptophan residues are located, which could be sensitive against singlet oxygen. Surprisingly, however, it has been found that only those species are protected, in which a second saccharide molecule is bound to the protein, probably at subsite E at the enzymatic center, where no sensitive amino acid side chains are located.

On the mechanism of the acridine orange sensitized photodynamic inactivation of lysozyme. I. Basic kinetics.

The kinetics of the photodynamic desactivation of lysozyme in presence of acridine orange as the sensitizer have been investigated in detail varying oxygen, protein, dye concentration, ionic strength and pH value. The kinetics can be approximately described as an over all pseudo-first-order rate process. Changing the solvent from water to D2O or by quenching experiments in presence of azide ions it could be shown that the desactivation of lysozyme is caused exclusively by singlet oxygen. The excited oxygen occurs via the triplet state of the dye with a rate constant considerably lower than that to be expected for a diffusionally controlled reaction. Singlet oxygen react chemically (desactivation, k=2.9 X 10(7) m(-1) sec(-1)) and physically (quenching process, k=4.1 X 10(8) m(-1) sec(-1)) with the enzyme. The kinetical analysis shows that additional chemical reaction between singlet oxygen and lysozyme would have only little influence on the kinetics of the desactivation as long as their products would be enzymatically active and their kinetical constants would be less than about 1 X 10(8) m(-1) sec(-1)).